Perivascular Adventitial Fibroblast Specialization Accompanies T Cell Retention in the Inflamed Human Dermis.

Perivascular accumulation of lymphocytes can be a prominent histopathologic feature of various human inflammatory skin diseases. Select examples include systemic sclerosis, spongiotic dermatitis, and cutaneous lupus. Although a large body of work has described various aspects of the endothelial and vascular smooth muscle layers in these diseases, the outer adventitial compartment is poorly explored. The goal of the current study was to characterize perivascular adventitial fibroblast states in inflammatory human skin diseases and relate these states to perivascular lymphocyte accumulation. In normal skin, adventitial fibroblasts are distinguished by CD90 expression, and dense perivascular lymphocytic infiltrates are uncommon. In systemic sclerosis, this compartment expands, but lymphocyte infiltrates remain sparse. In contrast, perivascular adventitial fibroblast expression of VCAM1 is upregulated in spongiotic dermatitis and lupus and is associated with a dense perivascular T cell infiltrate. VCAM1 expression marks transitioned fibroblasts that show some resemblance to the reticular stromal cells in secondary lymphoid organs. Expanded adventitial compartments with perivascular infiltrates similar to the human settings were not seen in the inflamed murine dermis. This species difference may hinder the dissection of aspects of perivascular adventitial pathology. The altered perivascular adventitial compartment and its associated reticular network form a niche for lymphocytes and appear to be fundamental in the development of an inflammatory pattern.

Developing dashboards for performance improvement in cytopathology.

INTRODUCTION: Cytopathology has well-defined and objective quality metrics for monitoring the performance of cytopathologists (CPs) and cytotechnologists (CTs). We transformed these metrics into dashboards for real-time visualization and on-demand feedback. METHODS: Dashboards were constructed with data from the previous 10 calendar years using the software Tableau. The dashboards for CPs were designed to display 2 gynecologic metrics and 1 nongynecologic metric: the ASCUS:SIL ratio, the percentage of high-risk human papillomavirus (HPV)-positive ASCUS interpretations (HPV+ ASCUS rate), and the proportion of AUS/FLUS thyroid interpretations. CT dashboards were designed to include these plus 2 others: the percentage of Papanicolaou tests referred for CP review and the percentage of Papanicolaou tests interpreted as unsatisfactory. Established professional benchmarks or standard deviations were used to set color-coded "goal," "borderline," and "attention" zones. RESULTS: Personal dashboards were successfully developed and implemented for CPs and CTs in the laboratory, with results that are automatically updated every week, requiring minimal curation. Each CP and CT has a unique link that allows them access to their results at any time. Color-coded displays show the individual their quality metrics over the past 10 years, with a snapshot of data from the past 3 months. The laboratory director has a unique link that allows the director access to results for each individual and the laboratory in aggregate. CONCLUSIONS: Personalized dashboards enable individuals to access their performance metrics on demand and examine recent performance as well as patterns over time. This facilitates self-motivation to improve performance and adhere to professional benchmarks.

Use of Deep Learning to Develop and Analyze Computational Hematoxylin and Eosin Staining of Prostate Core Biopsy Images for Tumor Diagnosis.

Importance: Histopathological diagnoses of tumors from tissue biopsy after hematoxylin and eosin (H&E) dye staining is the criterion standard for oncological care, but H&E staining requires trained operators, dyes and reagents, and precious tissue samples that cannot be reused. Objectives: To use deep learning algorithms to develop models that perform accurate computational H&E staining of native nonstained prostate core biopsy images and to develop methods for interpretation of H&E staining deep learning models and analysis of computationally stained images by computer vision and clinical approaches. Design, Setting, and Participants: This cross-sectional study used hundreds of thousands of native nonstained RGB (red, green, and blue channel) whole slide image (WSI) patches of prostate core tissue biopsies obtained from excess tissue material from prostate core biopsies performed in the course of routine clinical care between January 7, 2014, and January 7, 2017, at Brigham and Women's Hospital, Boston, Massachusetts. Biopsies were registered with their H&E-stained versions. Conditional generative adversarial neural networks (cGANs) that automate conversion of native nonstained RGB WSI to computational H&E-stained images were then trained. Deidentified whole slide images of prostate core biopsy and medical record data were transferred to Massachusetts Institute of Technology, Cambridge, for computational research. Results were shared with physicians for clinical evaluations. Data were analyzed from July 2018 to February 2019. Main Outcomes and Measures: Methods for detailed computer vision image analytics, visualization of trained cGAN model outputs, and clinical evaluation of virtually stained images were developed. The main outcome was interpretable deep learning models and computational H&E-stained images that achieved high performance in these metrics. Results: Among 38 patients who provided samples, single core biopsy images were extracted from each whole slide, resulting in 102 individual nonstained and H&E dye-stained image pairs that were compared with matched computationally stained and unstained images. Calculations showed high similarities between computationally and H&E dye-stained images, with a mean (SD) structural similarity index (SSIM) of 0.902 (0.026), Pearson correlation coefficient (PCC) of 0.962 (0.096), and peak signal to noise ratio (PSNR) of 22.821 (1.232) dB. A second cGAN performed accurate computational destaining of H&E-stained images back to their native nonstained form, with a mean (SD) SSIM of 0.900 (0.030), PCC of 0.963 (0.011), and PSNR of 25.646 (1.943) dB compared with native nonstained images. A single blind prospective study computed approximately 95% pixel-by-pixel overlap among prostate tumor annotations provided by 5 board certified pathologists on computationally stained images, compared with those on H&E dye-stained images. This study also used the first visualization and explanation of neural network kernel activation maps during H&E staining and destaining of RGB images by cGANs. High similarities between kernel activation maps of computationally and H&E-stained images (mean-squared errors <0.0005) provide additional mathematical and mechanistic validation of the staining system. Conclusions and Relevance: These findings suggest that computational H&E staining of native unlabeled RGB images of prostate core biopsy could reproduce Gleason grade tumor signatures that were easily assessed and validated by clinicians. Methods for benchmarking, visualization, and clinical validation of deep learning models and virtually H&E-stained images communicated in this study have wide applications in clinical informatics and oncology research. Clinical researchers may use these systems for early indications of possible abnormalities in native nonstained tissue biopsies prior to histopathological workflows.

Utility of Histologic and Histochemical Screening for 16S Ribosomal RNA Gene Sequencing of Formalin-Fixed, Paraffin-Embedded Tissue for Bacterial Endocarditis.

OBJECTIVES: 16S ribosomal RNA (rRNA) sequencing is a powerful but expensive tool for the identification of bacteria in culture-negative endocarditis. Histologic criteria to screen formalin-fixed, paraffin-embedded (FFPE) specimens for testing are evaluated. METHODS: Sixty-eight cases of infective endocarditis and controls were histologically reviewed and analyzed by 16S rRNA gene sequencing. RESULTS: Sequencing identified a specific pathogenic organism in 33 (49%) of 68 cases with acute inflammation and in 0 of 10 controls (P = .004). Visualization of organisms by Gram or Grocott methenamine silver stains had the strongest association with positive sequencing, while antibiotic treatment effect and acid decalcification decreased sensitivity. Molecular identifications were concordant with blood culture results in 90% of the cases, and a positive sequencing result was obtained in approximately half of the cases with negative valve cultures. CONCLUSIONS: Histologic screening criteria are extremely helpful for identifying cases likely to be positive by molecular testing and can provide significant cost savings in filtering out low-yield specimens.

Iron in neurodegenerative disorders of protein misfolding: a case of prion disorders and Parkinson's disease.

SIGNIFICANCE: Intracellular and extracellular aggregation of a specific protein or protein fragments is the principal pathological event in several neurodegenerative conditions. We describe two such conditions: sporadic Creutzfeldt-Jakob disease (sCJD), a rare but potentially infectious and invariably fatal human prion disorder, and Parkinson's disease (PD), a common neurodegenerative condition second only to Alzheimer's disease in prevalence. In sCJD, a cell surface glycoprotein known as the prion protein (PrP(C)) undergoes a conformational change to PrP-scrapie, a pathogenic and infectious isoform that accumulates in the brain parenchyma as insoluble aggregates. In PD, α-synuclein, a cytosolic protein, forms insoluble aggregates that accumulate in neurons of the substantia nigra and cause neurotoxicity. RECENT ADVANCES: Although distinct processes are involved in the pathogenesis of sCJD and PD, both share brain iron dyshomeostasis as a common associated feature that is reflected in the cerebrospinal fluid in a disease-specific manner. CRITICAL ISSUES: Since PrP(C) and α-synuclein play a significant role in maintaining cellular iron homeostasis, it is important to understand whether the aggregation of these proteins and iron dyshomeostasis are causally related. Here, we discuss recent information on the normal function of PrP(C) and α-synuclein in cellular iron metabolism and the cellular and biochemical processes that contribute to iron imbalance in sCJD and PD. FUTURE DIRECTIONS: Improved understanding of the relationship between brain iron imbalance and protein aggregation is likely to help in the development of therapeutic strategies that can restore brain iron homeostasis and mitigate neurotoxicity.

Brain iron homeostasis: from molecular mechanisms to clinical significance and therapeutic opportunities.

Iron has emerged as a significant cause of neurotoxicity in several neurodegenerative conditions, including Alzheimer's disease (AD), Parkinson's disease (PD), sporadic Creutzfeldt-Jakob disease (sCJD), and others. In some cases, the underlying cause of iron mis-metabolism is known, while in others, our understanding is, at best, incomplete. Recent evidence implicating key proteins involved in the pathogenesis of AD, PD, and sCJD in cellular iron metabolism suggests that imbalance of brain iron homeostasis associated with these disorders is a direct consequence of disease pathogenesis. A complete understanding of the molecular events leading to this phenotype is lacking partly because of the complex regulation of iron homeostasis within the brain. Since systemic organs and the brain share several iron regulatory mechanisms and iron-modulating proteins, dysfunction of a specific pathway or selective absence of iron-modulating protein(s) in systemic organs has provided important insights into the maintenance of iron homeostasis within the brain. Here, we review recent information on the regulation of iron uptake and utilization in systemic organs and within the complex environment of the brain, with particular emphasis on the underlying mechanisms leading to brain iron mis-metabolism in specific neurodegenerative conditions. Mouse models that have been instrumental in understanding systemic and brain disorders associated with iron mis-metabolism are also described, followed by current therapeutic strategies which are aimed at restoring brain iron homeostasis in different neurodegenerative conditions. We conclude by highlighting important gaps in our understanding of brain iron metabolism and mis-metabolism, particularly in the context of neurodegenerative disorders.

Prion protein regulates iron transport by functioning as a ferrireductase.

Prion protein (PrPC) is implicated in the pathogenesis of prion disorders, but its normal function is unclear. We demonstrate that PrPC is a ferrireductase (FR), and its absence causes systemic iron deficiency in PrP knock-out mice (PrP-/-). When exposed to non-transferrin-bound (NTB) radioactive-iron (59FeCl3) by gastric-gavage, PrP-/- mice absorb significantly more 59Fe from the intestinal lumen relative to controls, indicating appropriate systemic response to the iron deficiency. Chronic exposure to excess dietary iron corrects this deficiency, but unlike wild-type (PrP+/+) controls that remain iron over-loaded, PrP-/- mice revert back to the iron deficient phenotype after 5 months of chase on normal diet. Bone marrow (BM) preparations of PrP-/- mice on normal diet show relatively less stainable iron, and this phenotype is only partially corrected by intraperitoneal administration of excess iron-dextran. Cultured PrP-/- BM-macrophages incorporate significantly less NTB-59Fe in the absence or presence of excess extracellular iron, indicating reduced uptake and/or storage of available iron in the absence of PrPC. When expressed in neuroblastoma cells, PrPC exhibits NAD(P)H-dependent cell-surface and intracellular FR activity that requires the copper-binding octa-peptide-repeat region and linkage to the plasma membrane for optimal function. Incorporation of NTB-59Fe by neuroblastoma cells correlates with FR activity of PrPC, implicating PrPC in cellular iron uptake and metabolism. These observations explain the correlation between PrPC expression and cellular iron levels, and the cause of iron imbalance in sporadic-Creutzfeldt-Jakob-disease brains where PrPC accumulates as insoluble aggregates.